The Factor VIII complex has two distinct biologic functions: coagulant activity and a role in primary hemostasis. The analysis of Factor VIII deficiency diseases, classic hemophilia and von Willebrand's disease, have contributed to the understanding that Factor VIII is a complex of two components. The Factor VIII:c procoagulant protein (antihemophilic factor) and the Factor VIII related antigen (von Willebrand factor,VWF) are under separate genetic control, have distinct biochemical and immunologic properties, and have unique and essential physiologic functions.
The Factor VIII:c molecule is an important regulatory protein in the blood coagulation cascade. After activation by thrombin, it accelerates the rate of Factor X activation by Factor IXa, eventually leading to the formation of the fibrin clot. Deficiency of Factor VIII:c (classic hemophilia) is an X-linked chromosomal disorder that has been a major source of hemorrhagic morbidity and mortality in affected males. Treatment usually consists of frequent transfusions with blood products. The latter has resulted in a high incidence of infectious complications (such as various forms of hepatitis and acquired immunodeficiency disease) in the hemophiliac population.
The VWF molecule is an adhesive glycoprotein that plays a central role in platelet agglutination. It serves as a carrier for Factor VIII:c in plasma and facilitates platelet-vessel wall interactions. Discrete domains of VWF which bind to platelet receptor sites on glycoprotein 1b and on the glycoprotein IIb-IIIa complex, as well as binding sites on collagen have been noted. VWF is made up of multiple, probably identical, subunits each of about 230,000 daltons. VWF is synthesized in endothelial cells and megakaryocytes. In the plasma it exists as high molecular weight multimers ranging from 5.times.105 to 10.sup.7 daltons. Von Willebrand Factor contains 5-6% complex carbohydrate, which appears important in the molecule's ability to bind platelets. A variety of abnormalities in VWF activity can result in Von Willebrand's disease. The disorder is generally inherited in autosomal dominant fashion and may affect as many as one in 2000 individuals. Mild forms of the disease frequently go undiagnosed, whereas severely affected patients may require frequent blood product support with its associated risks.
Recently, the isolation of the genes for both Factor VIII:c and Von Willebrand Factor have made possible the production of recombinant factor VIII:c and VWF preparations, respectively, which are essentially free of contaminating viruses (Toole et al., 1984, Nature 312:342; Wood et al., 1984, Nature 312:330; Lynch et al., 1985, Cell 41:49-56; Ginsberg et al., 1985, Science 228:1401-1406). The production of Factor VIII:c or analogs thereof through recombinant DNA technology has been achieved utilizing mammalian cells transfected or transformed with appropriate expression vectors containing DNA encoding Factor VIII:c or the analogs thereof. Primary concerns for the synthesis of recombinant Factor VIII:c-type proteins include (i) the yield of recombinant protein obtainable from the culture medium, (ii) the stability of the recombinant protein so produced, (iii) the efficiency and cost of purification of the protein and (iv) the overall cost of producing purified recombinant protein.
For best results, we have heretofore typically cultured cells producing FVIII:c-type protein in media containing mammalian serum, e.g. conventional preparations of fetal bovine serum, in amounts of about 10% serum by volume relative to total media volume. (For the sake of simplicity, all serum concentrations hereinafter are expressed as a volume % of total media.) We have found that in the absence of serum supplements both the yield and stability of the recombinant FVIII:c suffer significantly. However, the cost of serum and the added inconvenience and expense in purification resulting from the addition of serum to the media rendered the use of serum an undesireable necessity and the wide scale use of recombinant FVIII:c a commercially less attractive alternative to natural FVIII:c purified from plasma. Interestingly, despite the great excitement in the medical and pharmaceutical communities over the clinical potential of recombinant FVIII:c, we are aware of no reports heretofore of the above-mentioned serum dependence, its biochemical basis or methods to circumvent it.
After extensive experimental modifications of media for FVIII:c-producing cells we have surprisingly found a method for producing higher yields of stable FVIII:c-type proteins (as described hereinafter) even when using media containing reduced amounts of serum (e.g., semi-defined media, containing .about.1% serum) or essentially serum-free media (defined media). We have found that host cells producing FVIII:c-type proteins produce recoverable, stable FVIII:c-type proteins in semi-defined and defined media in yields at least comparable, and in some cases superior, to those obtained in the presence of 10% serum if the semi-defined or defined media contains a suitable amount of a hydrophobic substance such as VWF or certain phospholipids. We have further found that FVIII:c-type proteins produced in semi-defined or defined media lacking such supplements typically exhibit dramatic instability and are recoverable in extremely low yield if at all.
Evidence to date suggests either that VWF may have a stabilizing effect in vivo on the Factor VIII:c in plasma, or that the VWF can ellicit the in vivo release from storage depots or stimulate the in vivo synthesis and/or secretion of Factor VIII:c (Weiss, H. J. et al., 1977,J. Clin. Invest. 60: 390-404). It has also been suggested that thrombin-activated Factor VIII (derived from natural, human FVIII) is stabilized by phospholipids, presumably with respect to thrombin-mediated degradation. See Andersson and Brown, 1981, Biochem. J. 200:161-167. However, to our knowledge there is no suggestion in the prior art as to any possible effect(s) of VWF or phospholipid on the in vitro production of Factor VIII:c-type proteins (where, for example, thrombin is substantially absent) or any suggestion of media supplements for the production of Factor VIII:c comprising VWF or a VWF-type protein and/or phospholipids either substantially free from the complex mixture of components present in mammalian serum or in concentrations higher than afforded by 10% serum supplements in accordance with this invention. It should be noted that mammalian serum contains VWF. As a point of reference, conventional media for mammalian cells which contains about 10% serum, contains about lug VWF/ml media.